Regulated neuronal death by apoptosis is a key part of normal brain development. Unfortunately, neuronal apoptosis is also triggered after brain injury or in neurodegenerative disease, where it contributes to the loss of neurons and the resulting neurological deficits. Thus, the neuronal apoptosis pathway is of physiological and pathological importance. Recent studies have reported an age-dependent decline in the incidence of apoptosis in the brain. However, the mechanisms of these changes are not well studied, and its implications on how apoptosis is activated in neuropathological situations in young versus adult neurons are unclear. Our long-term objectives are to understand the molecular mechanisms by which apoptosis is regulated in neurons, with a specific interest in identifying age-related changes in this pathway. Our focus is on the mechanism of activation of caspase proteases, which are the central executioners of apoptosis. A major pathway of caspase activation involves the mitochondrial release of cytochrome c and subsequent formation of the Apaf-1-dependent apoptosome complex. Our results have identified distinct age-dependent changes in which the cytochrome c-dependent caspase activation pathway becomes increasingly inhibited in neurons. For example, we find that caspase activation in neurons during early development is strictly and selectively regulated by XIAP. In Specific Aim 1, we will examine how XIAP's strict control of caspases is overcome to permit caspase activation in physiological (NGF deprivation) and pathological (ER stress) models of sympathetic neuronal apoptosis. We will also examine whether XIAP-deficient neurons are more vulnerable to neuronal injury in vitro and in vivo. We will examine this in neurodegeneration models of mutant superoxide dismutase 1 (SOD1) overexpression and neonatal hypoxia-ischemia. At later stages of development, we find that neurons engage additional mechanisms to inhibit apoptosis. In Specific Aim 2, we will focus on identifying the posttranslational mechanism that inactivates Apaf-1 in neurons during the late period of cerebellar development. Finally, as neurons mature, they appear to turn off the expression of Apaf-1 and caspase-3 altogether. In Specific Aim 3 we will test whether the cell cycle- related protein E2F1, which is known to be expressed in dying neurons, is important for restoring Apaf-1 expression and permitting cytochrome c-mediated apoptosis in mature neurons. Understanding the mechanism by which apoptosis is regulated in neurons is clinically important for identifying appropriate drug targets that would effectively prevent neuronal death after injury or disease.